AIM:
To evaluate attenuating properties of N-acetylcysteine (NAC) on
oxidative stress and liver pathology in rats with non-alcoholic
steatohepatitis (NASH).

METHODS:
Male Sprague-Dawley rats were randomly divided into three groups.
Group 1 (control, n = 8) was free accessed to regular dry rat chow
(RC) for 6 wk. Group 2 (NASH, n = 8) was fed with 100% fat diet for
6 wk. Group 3 (NASH + NAC20, n = 9) was fed with 100% fat
diet plus 20 mg/kg per day of NAC orally for 6 wk. All rats were
sacrificed to collect blood and liver samples at the end of the
study.

RESULTS:
The levels of total glutathione (GSH) and hepatic malondialdehyde (MDA)
were increased significantly in the NASH group as compared with the
control group (GSH; 2066.7 ± 93.2 vs 1337.5 ± 31.5 mmol/L
and MDA; 209.9± 43.9 vs 3.8 ±1.7 mmol/g
protein, respectively, P < 0.05). Liver histopathology from group 2
showed moderate to severe macrovesicular steatosis, hepatocyte
ballooning, and necroinflammation. NAC treatment improved the level
of GSH (1394.8 ± 81.2 mmol/L,
P < 0.05), it did not affect MDA (150.1 ± 27.0 mmol/g
protein), but led to a decrease in fat deposition and
necroinflammation.

Non-alcoholic steatohepatitis (NASH) is a liver
disease characterized by macrovesicular steatosis, hepatocyte
necrosis, inflammation, Mallory bodies, and fibrosis[1].
NASH is closely associated with the metabolic or insulin resistance
syndrome[2]. This is a cluster of disorders, such as
obesity, diabetes mellitus, dyslipidemia, arteriosclerosis, and
hypertension, with insulin resistance as a common feature[3].
In initial phases, during which fat accumulates in the liver, no
clinical symptoms are evident. In advanced stages, fibrosis is
detectable, which might progress into cirrhosis in some patients[4].

There are many models of NASH-like liver injuries in
animals as the genetic model of ob/ob mice[5], the
methionine and choline deficient diet model[6,7], and a
model with high-fat liquid diet in which 71% of energy is derived
from fat, 11% from carbohydrates, and 18% from protein[8].

Oxidative stress is believed to play an important
role in pathogenesis of NASH. It is likely involved in the
progression of disease from steatosis to NASH and potentially
cirrhosis. It has been shown that chronic oxidative stress,
generated through the oxidation of cytotoxic free fatty acids, can
lead to upregulation of cytokines[9], induction of the
liver cytochrome P450 enzyme 2E1 (CYP2E1), and depletion of hepatic
antioxidant concentration[10]. In addition, enhanced
lipid peroxidation leads to the generation of byproducts, such as
4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), which have been
shown to further stimulate cytokine production. They are involved in
hepatic stellate cell activation[11], fibrogenesis, and
enhanced extracellular matrix protein deposition.

According to the concepts of pathogenesis of NASH,
these might make a wise basis for the use of antioxidants or drugs
that could protect hepatocytes from oxidative stress. N-acetylcysteine
(NAC) is a glutathione precursor which increases glutathione levels
in hepatocytes[12]. Increased glutathione levels, in
turn, limit the production of reactive oxygen species (ROS) which
cause hepatocellular injury[13]. Oral NAC treatment (1
g/d) of 11 NASH patients for 3 mo was demonstrated to improve liver
function test significantly at the end of treatment period[12].
In a controlled study, NAC (600 mg/d)
was administered to NASH patients for 4 wk, and a significant
improvement in aminotransferase levels was found[14].
Although NAC was shown to improve liver function test in NASH
patients, the mechanism remained unclear. Treatment of NASH with
diet or diet plus NAC could attenuate oxidative stress as well as
improve biochemical parameters and liver histopathology. However,
the result of addition of NAC is not better than diet treatment
alone[15]. Therefore, this study was conducted to
determine the effects of NAC on oxidative stress and liver pathology
in a rat model of 100% fat diet induced NASH[16].

MATERIALS AND METHODS

Animal preparation

This study was approved by the Ethics Committee of
the Faculty of Medicine, Chulalongkorn University, Bangkok,
Thailand. Male Sprague-Dawley rats weighing 220-260 g from the
National Laboratory Animal Center, Mahidol University, Salaya,
Nakorn Pathom were used. The animals were allowed to rest for a week
after arrival at the Animal Center, Department of Physiology,
Faculty of Medicine, Chulalongkorn University. They were kept at a
controlled temperature of 25 ± 1℃
under standard conditions (12 h dark: 12 h light cycle), fed with
regular dry rat chow ad libitum, and had freely access to drinking
water.

All rats were weighed weekly. They were sacrificed to
collect blood, serum, and liver samples at the end of the study, 20
h after the last NAC treatment. The diagram of the experiment was
shown as follow.

At the end of the study, all rats were anaesthetized
using intraperitoneal injection of an overdose (45 mg/kg) of sodium
pentobarbital, and the abdominal walls were opened. Blood was drawn
by cardiac puncture for total glutathione assay and biochemical
assay. The livers were excised quickly and cleaned in iced-cold NSS.
One lobe of the liver was collected for MDA measurement, the
remaining liver was fixed in 40 g/L formaldehyde solution for
histological examination.

Total glutathione determination

Total glutathione levels were quantified using
Cayman’s
GSH assay kit. This assay uses glutathione reductase for
determination of glutathion. The sulfhydryl group of glutathion
reacts with DTNB (5, 5’-dithiobis-2-nitrobenzoic acid, Ellman’s
reagent) and produces a yellow colored 5-thio-2-nitrobenzoic acid (TNB).
The mixed disulfide, GSTNB (between glutathion and TNB) that is
concomitantly produced, is reduced by glutathione reductase to
recycle glutathion and to produce more TNB. The rate of TNB
production is directly proportional to this recycling reaction which
is in turn directly proportional to the concentration of glutathionn
in the sample. Measurement of the absorbance of TNB at 405 nm
provides an accurate estimation of glutathion in the sample.

Hepatic malondialdehyde (MDA) determination

One lobe of the liver was removed and weighed. One
gram of the tissue was placed in a test tube containing 2.25 mL
homogenization buffer (11.5 g/L KCl) and homogenized in an ice box
using a homogenizer at a rotational speed of 12 000 r/min for 1 min.
MDA was quantified by using the thiobarbituric acid reaction as
described by Ohgawa et al[17]. MDA levels in the
samples were determined the linear regression equation from a
standard curve. The content of lipid peroxide is expressed as nmol
of MDA/g of wet weight, and the total protein was determined by the
Lowry method[18] to correct the MDA level which is
expressed in terms of mmol/g
protein.

Histopathological examination

The remaining liver samples were fixed in 40 g/L
formaldehyde solution at room temperature. They were processed by
standard methods. Briefly, tissues were embedded in paraffin,
sectioned at 5 mm,
stained with HE, and then picked up on glass slides for light
microscopy. An experienced pathologist blinded to the experiment
evaluated all samples. All fields in each section were examined for
grading of steatosis and necroinflammation according to the criteria
described by Brunt et al[19].

The severity of steatosis was scored on the basis of
the extent of involved parenchyma as 1 if fewer than 33% of the
hepatocytes were affected, as 2 if 33%-66% of the hepatocytes were
affected, as 3 if more than 66% of the hepatocytes were affected,
and as 0 if no hepatocytes were affected.

The data were expressed as mean ± SEM using the SPSS
version 11.5 for Windows program. Statistical comparisons between
groups were analyzed by ANOVA and post hoc comparisons were done
with Bonferroni correction. P < 0.05 were considered significant.

RESULTS

Body mass and general condition

The body mass at 6 wk of the NASH group and NASH +
NAC20 group were decreased compared to the control (197.0
± 8.1 g, 207.8 ± 6.9 g vs 438.4 ± 9.7 g, P < 0.05).
Despite weight loss, the general condition of 100% fat diet-fed rats
remained good throughout the observation periode. After the first 6
wk, rats were fed with regular dry rat chow for additional 4 wk. The
body mass was significantly increased in all groups (Table
1).

Serum biochemical parameters

Serum biochemical parameters in the control and the
experimental groups are given in Table 1. Serum AST and ALT
activities decreased significantly in the NASH group when compared
to the control group (AST; 53.7 ± 9.3 U/L vs 86.8 ± 4.3 U/L,
ALT; 23.0 ± 1.9 U/L vs 40.1 ± 2.4 U/L, P < 0.05).
Serum ALT but not AST activity returned to control levels in the
NASH + NAC20 group (ALT 25.4 ± 5.7 U/L; AST 65.6 ± 8.7
U/L). Serum cholesterol was significantly higher in the NASH group
and NASH + NAC20 group than that in the control group
(94.8 ± 3.1 g/L, 91.4 ± 3.5 g/L vs 71.8 ± 1.8 g/L, P <
0.05), whereas there were
no significant differences in serum triglycerides (Table 1).

MDA was elevated significantly in the NASH group when
compared to the control group (209.9 ± 43.8 mmol/g
protein vs 3.8 ± 1.7 mmol/g
protein, P < 0.05). There was no statistical significant
difference in MDA levels in NASH + NAC20 group (150.1 ±
27.0 mmol/g
protein).

Histopathological examination

Liver sections from rats fed with the regular dry rat
chow had normal morphological appearance. In the NASH group, all
animals developed moderate to severe macrovesicular steatosis,
hepatocyte ballooning, mild to moderate inflammation, and
regeneration of hepatocytes (Table
2). NAC treatment improved steatosis and necroinflammation
scores in animals of the NASH + NAC20 group when compared
with the NASH group (Figure
1).

DISCUSSION

Histopathology of NASH is similar to that of
ethanol-induced hepatitis with the presence of macrovesicular
steatosis, hepatocyte ballooning, necroinflammation, Mallory bodies,
and fibrosis[1]. To study the pathogenesis of or
therapeutic options for NASH, there are many models that can be used
including a genetic model (obese rats), a model of methionine and
choline deficient diet, a model of high fat liquid diet, and a 100%
fat diet[5-8,16]. In this study, 100% fat diet was chosen
to induce NASH in Sprague-Dawley rats as this procedure is fast,
easy, and provides a comparable pattern of pathological changes as
in humans although this model represents malnutrition induced
steatohepatitis.

By feeding rats with 100% fat diet, the hepatic
lesions of NASH were apparent within 6 wk. Histopathological
examination showed macrovesicular steatosis, hepatocyte ballooning,
Mallory bodies, and mild to moderate inflammation. One hundred
percent fat diet caused mobilization of free fatty acid (FFA) from
adipose tissue and transport into hepatocytes. In this condition,
the liver failed to synthesize apolipoprotein that is required for
packaging and exporting fat from the liver, triglycerides (TG) thus
accumulate in the liver[20]. b-oxidation
of FFA in hepatocytes produces reactive oxygen species (ROS) which
activate lipid peroxidation[21]. ROS and lipid
peroxidation cause direct damage to hepatocytes by disrupting
membranes, protein, and DNA[22,23].
Hepatocyte damage and lipid peroxidation products induce an
inflammatory response.

AST
and ALT are useful screening tests for detecting liver injury[24].
They are found in hepatocytes and can not diffuse out of the cells
in the physiological condition. When the hepatocyte is injured,
plasma membrane can be disrupted and the leakage through
extracellular fluid of the enzyme occurs where they can be detected
at abnormal levels in the serum[25]. AST and ALT
activities have been found to be increased in NASH rats[10,26-29].
In contrast, AST and ALT activities decreased significantly with 6
wk of 100% fat diet in this study. The decreased serum transaminases
may be due to poor nutrition or hepatocyte death. Rats fed with 100%
fat diet derived main energy from fat, when there were low in
vitamin and mineral contents. The decreased AST and ALT levels were
probably due to nutritional deficiency of pyridoxal phosphate which
is a cofactor for both AST and ALT to catalyze the transfer of the a
amino group from aspartate or alanine to a-ketoglutarate with made
the release of pyruvate, oxaloacetate, and glutamate[24].
In addition, oxidative stress condition may be a cause of hepatocyte
death, therefore, aminotransferases can not be produced.

In
100% fat diet-fed rats, body mass decreased significantly (P
< 0.05) as compared to the control group. While serum cholesterol
significantly increased, serum TG level was unchanged. Feeding with
100% fat diet for 6 wk caused a loss of body mass that may be due to
a metabolic imbalance of carbohydrate, protein, and fat. Moreover,
100% fat diet contained highly saturated fat which may increase
blood cholesterol concentration by 15% to 25%[30]. This
result was from an increase of fat deposition in the liver which
then provides the increased quantities of acetyl CoA in the liver
cell for production of cholesterol[30]. The increased
cholesterol was found in this experiment and had been observed in
another study that used 10% lard oil and 2% cholesterol supplement
adding into the standard diet[30].

FFA
causes oxidative stress that has the potential to induce NASH[2].
FFA in the body is increased and this is associated with state of
starvation[2]. Stored FFA can be mobilized from adipose
tissue through lipolysis[2]. FFA metabolism increases the
production of ROS which activated lipid peroxidation. Consequences
are the disruption of membranes and the production of reactive
metabolites such as MDA[21]. This study found high
hepatic MDA levels in 100% fat-diet fed rats in accordance with
studies by others[26-29]. Glutathione is the major
intracellular non-protein antioxidant and plays a crucial role in
the detoxification of free radicals[31,32]. Serum level
of glutathione was increased in patients with NASH[33].
Similarly in this experiment, an increasing in total glutathione in
whole blood with 100% fat diet feeding could be explained by
compensatory protection mechanism against oxidative stress.

NAC
is a thiol compound that acts directly as free radical scavenger and
as a precursor of reduced glutathione[34]. Therefore,
treatment with 20 mg/kg of NAC improved the total glutathione level
to normal level in NASH + NAC20 group and improved
necroinflammation score. Because of some limitations of our study,
such as dose of NAC, time for treatment, and the number of animals,
the effect of NAC on reducing hepatic MDA level remained unclear. In
our previous study, diet treatment alone and diet plus NAC groups,
total glutathione, serum AST, ALT, cholesterol, TG, and hepatic MDA
returned to normal levels as in the control group. In addition, the
pathological changes of liver in these groups were improved[15].
These results emphasized how crucial the nutritional composition of
the diet is. Good proportion of nutrients (i.e., carbohydrate,
lipid, and protein) is essential for growth and maintenance. These
nutrients supply energy, promote growth, repair body tissues, and
regulate metabolic processes[35].

In
conclusion, feeding with 100% fat diet for 6 wk induced
macrovesicular steatosis, hepatocyte ballooning, and inflammation in
rats similar to histopathology of NASH. Treatment with NAC in NASH
could improve oxidative stress and liver histopathology.

COMMENTS

Background

Non-alcoholic steatohepatitis (NASH), in advanced stages, can cause
liver fibrosis, eventually progressing to cirrhosis in some
patients. Oxidative stress is believed to play an important role in
pathogenesis of NASH. N-acetylcysteine (NAC) is a glutathione
precursor which increases glutathione levels in hepatocytes.
Increased glutathione levels, in turn, limit the production of
reactive oxygen species (ROS) which cause hepatocellular injury that
could protect hepatocytes from oxidative stress.

Research frontiers

NAC
is a thiol compound that acts directly as free radical scavenger. In
the pathogenesis of NASH, prevention of oxidative stress could
protect hepatocytes from injury. The hotspots of this study indicate
that NAC treatment could attenuate oxidative stress and improve
liver histology in rats with NASH.

Innovations and breakthroughs

According to a previous report, oral NAC treatment of NASH patients
for several months was found to significantly improve
aminotransferase levels. However, the mechanism remained unclear.
This study is a novel and well conducted experimental study showing
the efficacy of NAC on improvement of total glutathion level and
hepatic MDA in rats with NASH. Furthermore, treatment with NAC
showed improvement in steatosis and necroinflammation.

NASH is a liver disease characterized by
macrovesicular steatosis, hepatocyte necrosis, inflammation, Mallory
bodies, and fibrosis. In initial phases, during which fat
accumulates in the liver, no clinical symptoms are evident. In
advanced stages, fibrosis is detectable, eventually progressing to
cirrhosis. NAC is a glutathione precursor which increases
glutathione levels in hepatocytes. Increased glutathione levels, in
turn, limit the production of ROS which cause hepatocellular injury.

Peer review

This
is an experimental work on a steatosis model in the rat, induced by
100% fat diet in which the co-administration of NAC protects against
fat induced liver injury. This is a very interesting and well
conducted experimental study showing the efficacy of NAC in
preventing biochemical and histological alterations secondary to a
fat rich diet.